Project Summary Both genetic and environmental factors conspire during early stages of autoimmune disease to break immune tolerance. Several risk alleles associated with an increased risk of developing systemic lupus erythematosus (SLE), and other autoimmune diseases, target the function of the Src-family tyrosine kinase Lyn. Lyn plays an important role in immune tolerance by initiating inhibitory signaling in autoreactive B cells, leading to a state of unresponsiveness or anergy. Important downstream phosphatases that mediate this inhibitory signaling are SHIP-1 and SHP-1. It is still unclear how Lyn facilitates the activation of SHIP-1 and SHP-1 in anergic B cells. Lyn phosphorylates specific motifs on inhibitory receptors (ITIM or ITSM) that enables these receptors to recruit and activate phosphatases. The aims of this proposal are to identify the Lyn-dependent inhibitory receptors required to activate SHIP-1 (Aim 1) and SHP-1 (Aim 2) in anergic B cells. We will use a novel technique, Inhibitory Receptor Trap (IRT), to identify these inhibitory receptors. IRT uses SH2 domains from SHIP-1 or SHP-1 to specifically isolate phosphorylated inhibitory receptors (indicating that the receptors are active). Isolated receptors will be identified by mass spectrometry. We will evaluate the relevance of identified receptors in anergic B cells from man and mouse. Understanding the molecular pathways by which inhibitory signaling circuits are activated in autoreactive B cells is important. Many risk alleles associated with an increased risk to develop autoimmune disease affect these inhibitory circuits. Delineating pathway relationships will allow for more accurate predictions of which risk alleles will act synergistically. This will enable us to identify individuals at heightened risk and will allow for earlier intervention and possible preventive care. Identified receptors are also potential novel targets for therapeutic intervention which may allow us to restore B cell tolerance.